2 Abstract Faulty vitamin D metabolism in children less than 12 months of age can lead to formation of the inactive 3-epi- 25 monohydroxy form. The resolution of 3-epimer from the active monohydroxy form by tandem mass spectrometry is not possible due to mostly identical fragmentation pattern of the two species. As a result, the two isomers should be separated chromatographically. The method described here resolves the critical pair within a short run time. Serum/plasma samples were treated with acetonitrile to precipitate the protein, followed by centrifugation. A small volume of the supernatant was injected on the LC column. The chromatographic separation is carried out by a high efficiency media that allowed for separation of the monohydroxy vitamin D3 isomers as well as separation of the 3-epi-25 monohydroxy epimer. A typical methanol and formic acid mobile phase combination starting with high organic concentration is used. The column is maintained at ambient temperature, ~22 C. The signal detection is carried out by a triple quadrupole mass spectrometer operating in multiple reactions monitoring (MRM) function. An atmospheric pressure ionization source operating in positive polarity and using high purity nitrogen gas produced the [M+H + - H2O] + precursor ions. The LOD for both 25-OH-Vit D3 and its 3-epimer were similar at 2.5 ng/ml. The method prescribed here provides excellent resolution of the monohydroxy vitamin D3 isomers within a short run time.

3 Introduction In recent years, vitamin D (Ergocalciferol, D2 and Cholecalciferol, D3) has been subject to increasing investigation for a range of potentially beneficial health effects. The measurement of Vitamin D metabolites, 25-hydroxy (25-OH) and 1α, 25-DiOH vitamin D (Vit D), is used as marker to determine vitamin D deficiency. Isomerization of 25-OH-Vit D produces 3-epi Vit D3 (conversion of α-oh to ß-OH), a diasteromeric form. The presence of the epimer was first reported in 2006 by Singh et al. In infants, a significant portion of the 25-OH- Vit D may be present as the epimeric form. Thus, in order to determine the accurate vitamin D status of such patients, it is necessary to be able to distinguish between the two diastereomeric forms. Historically, analysis of Vit D and its metabolites has been performed via immunoassays. However, there is some question as to the ability of immunoassays to discriminate between 25-OH-D3 and its epimer. Thus, the development of an LC/MS/MS analysis that can distinguish the 25-OH- Vit D metabolite from its epimeric form is greatly desired.

6 Chromatographic Media 2.6 µm Core-Shell Particle 0.35 µm Porous Shell 1.9 µm Solid Core Performance equivalent to or better than fully porous Can be used on conventional HPLC systems and UH- The final, optimized LC/MS/MS method for the separation and analysis of 25-OH-Vit D and its epimer was performed using a core-shell column - Kinetex 2.6 µm PFP. The core-shell Kinetex particle consists of a solid inner core surrounded by a layer of porous silica material. This unique core-shell structure can provide exceptionally high efficiency at relatively modest backpressure (compatible with a conventional HPLC system).

12 Sample Preparation A protein precipitation method was devised to establish a calibration curve from 2.5 to 100 ng/ml. Commercially available serum could not be used due to its high contents of the OH-Vit D3 AND 3-epi forms (Figure 5). Double charcoal-stripped human serum was tested and found to have lower than 2.5 ng/ml concentration of OH-D2/D3. Sample preparation was carried out with the below procedure: 30 µl Int Std (OH-D3-2H3) and 200 µl sample was treated with 400 µl precipitation reagent (5:2:1 Methanol/Acetonitrile/Zinc Sulfate) and vortexed briefly, 4-5 sec The mixture was centrifuged at rpm for 7 minute The supernatant was decanted into an autosampler vial and placed in the autosampler A linear fit with 1/x weighting factor was used for both analytes and showed an excellent calibration fit (Figures 6-7.)

15 Conclusions We have developed an assay using the Kinetex 2.6 µm PFP column that can accurately quantitate 25-OH-Vit D3 in the presence of its epimeric form using a simple water/methanol/formic acid mobile phase. This assay can also be used to quantitate 25-OH-D2, 25-OH-D3, and the 25-OH- D3 epimer with a total analysis time of less than 5 minutes.

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